Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site.

Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.

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A striking similarity in the organization of the E-selectin and beta interferon gene promoters

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6464-6475.1994 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6464-6475 ◽

Cited By ~ 1

Author(s):

M Z Whitley ◽

D Thanos ◽

M A Read ◽

T Maniatis ◽

T Collins

Keyword(s):

Gene Expression ◽

Binding Sites ◽

Striking Similarity ◽

Tnf Alpha ◽

Gene Promoters ◽

Binding Studies ◽

Nf Kappa B ◽

Beta Interferon ◽

Interferon Gene

Transcription of the endothelial leukocyte adhesion molecule 1 (E-selectin or ELAM-1) gene is induced by the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). In this report, we identify four positive regulatory domains (PDI to PDIV) in the E-selectin promoter that are required for maximal levels of TNF-alpha induction in endothelial cells. In vitro DNA binding studies reveal that two of the domains contain novel adjacent binding sites for the transcription factor NF-kappa B (PDIII and PDIV), a third corresponds to a recently described CRE/ATF site (PDII), and a fourth is a consensus NF-kappa B site (PDI). Mutations that decrease the binding of NF-kappa B to any one of the NF-kappa B binding sites in vitro abolished cytokine-induced E-selectin gene expression in vivo. Previous studies demonstrated a similar correlation between ATF binding to PDII and E-selectin gene expression. Here we show that the high-mobility-group protein I(Y) [HMG I(Y)] also binds specifically to the E-selectin promoter and thereby enhances the binding of both ATF-2 and NF-kappa B to the E-selectin promoter in vitro. Moreover, mutations that interfere with HMG I(Y) binding decrease the level of cytokine-induced E-selectin expression. The organization of the TNF-alpha-inducible element of the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human beta interferon gene in that both promoters require NF-kappa B, ATF-2, and HMG I(Y). We propose that HMG I(Y) functions as a key architectural component in the assembly of inducible transcription activation complexes on both promoters.

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A striking similarity in the organization of the E-selectin and beta interferon gene promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6464 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6464-6475 ◽

Cited By ~ 136

Author(s):

M Z Whitley ◽

D Thanos ◽

M A Read ◽

T Maniatis ◽

T Collins

Keyword(s):

Gene Expression ◽

Binding Sites ◽

Striking Similarity ◽

Tnf Alpha ◽

Gene Promoters ◽

Binding Studies ◽

Nf Kappa B ◽

Beta Interferon ◽

Interferon Gene

Transcription of the endothelial leukocyte adhesion molecule 1 (E-selectin or ELAM-1) gene is induced by the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). In this report, we identify four positive regulatory domains (PDI to PDIV) in the E-selectin promoter that are required for maximal levels of TNF-alpha induction in endothelial cells. In vitro DNA binding studies reveal that two of the domains contain novel adjacent binding sites for the transcription factor NF-kappa B (PDIII and PDIV), a third corresponds to a recently described CRE/ATF site (PDII), and a fourth is a consensus NF-kappa B site (PDI). Mutations that decrease the binding of NF-kappa B to any one of the NF-kappa B binding sites in vitro abolished cytokine-induced E-selectin gene expression in vivo. Previous studies demonstrated a similar correlation between ATF binding to PDII and E-selectin gene expression. Here we show that the high-mobility-group protein I(Y) [HMG I(Y)] also binds specifically to the E-selectin promoter and thereby enhances the binding of both ATF-2 and NF-kappa B to the E-selectin promoter in vitro. Moreover, mutations that interfere with HMG I(Y) binding decrease the level of cytokine-induced E-selectin expression. The organization of the TNF-alpha-inducible element of the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human beta interferon gene in that both promoters require NF-kappa B, ATF-2, and HMG I(Y). We propose that HMG I(Y) functions as a key architectural component in the assembly of inducible transcription activation complexes on both promoters.

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Retinoic Acid Selectively Inhibits Lipopolysaccharide Induction of Tissue Factor Gene Expression in Human Monocytes

Blood ◽

10.1182/blood.v91.8.2857.2857_2857_2865 ◽

1998 ◽

Vol 91 (8) ◽

pp. 2857-2865 ◽

Cited By ~ 21

Author(s):

Paul Oeth ◽

Jin Yao ◽

Sao-Tah Fan ◽

Nigel Mackman

Keyword(s):

Retinoic Acid ◽

Tissue Factor ◽

Human Monocytes ◽

Monocytic Cells ◽

All Trans Retinoic Acid ◽

Tnf Α ◽

Activated Monocytes ◽

Tf Gene ◽

Factor Α ◽

Necrosis Factor

Expression of tissue factor (TF) by activated monocytes in several diseases leads to disseminated intravascular coagulation. Lipopolysaccharide (LPS)-induced monocyte TF expression is downregulated by the nuclear hormone all-trans retinoic acid (ATRA). In this study, we examined the mechanism by which ATRA inhibits monocyte TF expression. We show that ATRA selectively inhibited LPS induction of TF expression in human monocytes and monocytic THP-1 cells without affecting LPS induction of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8). Inhibition of TF expression occurred at the level of transcription as determined by nuclear run-on. ATRA did not significantly alter the binding or functional activity of the transcription factors c-Fos/c-Jun and c-Rel/p65, which are required for LPS induction of the TF promoter in monocytic cells. In contrast to the ATRA inhibition of the endogenous TF gene, LPS induction of the cloned TF promoter was not inhibited by ATRA in transiently transfected THP-1 cells. Our results demonstrate that ATRA selectively inhibited LPS-induced TF gene transcription in human monocytic cells by a mechanism that does not involve repression of AP-1– or NF-κB–mediated transcription.

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Tissue factor (TF) gene expression analysis in blood monocytes

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2003.tb04457.x ◽

2003 ◽

Vol 1 ◽

pp. P0541-P0541

Author(s):

J. Mueller ◽

J. Rox ◽

B. Poetzsch

Keyword(s):

Gene Expression ◽

Tissue Factor ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Blood Monocytes ◽

Tf Gene

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Pyrrolidine dithiocarbamate abrogates tissue factor (TF) expression by endothelial cells: evidence implicating nuclear factor-kappa B in TF induction by diverse agonists

Blood ◽

10.1182/blood.v86.2.436.bloodjournal862436 ◽

1995 ◽

Vol 86 (2) ◽

pp. 436-443 ◽

Cited By ~ 31

Author(s):

CL Orthner ◽

GM Rodgers ◽

LA Fitzgerald

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Nuclear Factor Kappa B ◽

Transcriptional Level ◽

Pyrrolidine Dithiocarbamate ◽

Dependent Manner ◽

Nuclear Factor ◽

Nf Kappa B ◽

Nuclear Factor Kappa ◽

Tf Gene

Tissue factor (TF), a 46-kD glycoprotein receptor for coagulation factors VII and VIIa, is expressed on the surface of endothelial cells in response to a variety of agonists and is thought to play an important role in initiating the thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. The induction of TF activity by lipopolysaccharide (LPS) is regulated, at least partially, at a transcriptional level and an LPS response element containing two activator protein-1 sites and a nuclear factor- kappa B (NF kappa B)-like site has been localized to the 5′ flanking region of the TF gene by transfection studies of TF promoter/reporter gene constructs. We have examined the effect of pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of the NF kappa B pathway on the expression of the endogenous TF gene in human umbilical vein endothelial cells (HUVEC). Preincubation of HUVEC for 60 minutes with PDTC inhibited LPS induction of TF activity on the cell surface in a dose-dependent manner, with 50% inhibition occurring at 10 mumol/L PDTC and 100% inhibition at higher concentrations (> or = 100 mumol/L). Furthermore, PDTC inhibited TF expression in response to tumor necrosis factor-alpha, interleukin-1 beta, and phorbol 12-myristate 13-acetate. The effect of PDTC was at the mRNA level, as seen by the complete abrogation of the large increase in TF mRNA observed in LPS-treated HUVEC. These results suggest that endothelial cell activation by diverse agonists initiates intracellular signaling events that converge upon a common pathway involving NF kappa B and, furthermore, that NF kappa B activation is an obligatory step induction of TF.

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Modelization of Blood-Borne Hypercoagulability in Myeloma: A Tissue-Factor-Bearing Microparticle-Driven Process

TH Open ◽

10.1055/s-0039-1700885 ◽

2019 ◽

Vol 03 (04) ◽

pp. e340-e347

Author(s):

Loula Papageorgiou ◽

Kutaiba Alhaj Hussen ◽

Sandrine Thouroude ◽

Elisabeth Mbemba ◽

Héléne Cost ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Tissue Factor ◽

Thrombin Generation ◽

Plasma Cells ◽

Annexin V ◽

Factor Vii ◽

Newly Diagnosed ◽

Normal Human ◽

Tf Gene

Abstract Introduction Hypercoagulability is a common blood alteration in newly diagnosed chemotherapy naïve patients with multiple myeloma. The identification of the procoagulant potential of cancer cells, which is principally related to tissue factor (TF) expression, attracts particular interest. The mechanisms by which myeloma plasma cells (MPCs) activate blood coagulation have been poorly investigated. Aim To identify the principal actors related with MPCs that boost thrombin generation (TG). Methods TF and annexin V expression by MPCs and MPC-derived microparticles (MPC-dMPs) was analyzed by flow cytometry. TF activity (TFa) and TF gene expression were also determined. TG in the presence of MPCs or MPC-dMPs was assessed with the calibrated automated thrombogram assay (CAT) in normal human PPP and in plasma depleted of factor VII or XII. TG was also assessed in plasma spiked with MPCs and MPC-dMPs. Results MPC-dMPs expressed approximately twofold higher levels of TF as compared with MPCs. The TFa expressed by MPC-dMPs was significantly higher compared with that expressed by MPCs. MPCs and MPC-dMPs enhanced TG of human plasma. TG was significantly higher with MPC-dMPs compared with MPCs. Conclusion MPCs indirectly induce blood-borne hypercoagulability through the release of MPC-dMPs rich in TF. Since MPCs, expressing low TFa, represent a weak procoagulant stimulus, the hypercoagulability at the microenvironment could be the resultant of MPC-dMPs rich in TF.

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MEG8 regulates Tissue Factor Pathway Inhibitor 2 (TFPI2) expression in the endothelium

10.21203/rs.3.rs-800527/v1 ◽

2021 ◽

Author(s):

Veerle Kremer ◽

Diewertje I. Bink ◽

Laura Stanicek ◽

Eva van Ingen ◽

Sarah Hilderink ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Function ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Umbilical Vein ◽

Regulation Of Gene Expression ◽

Underlying Mechanism ◽

Tissue Factor Pathway ◽

Non Coding Rnas

Abstract A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. The 14q32 locus contains many non-coding RNAs such as Maternally Expressed Gene 8 (MEG8). We observed an induction of this gene in ischemic heart disease. We investigated the role of MEG8 specifically in endothelial function as well as the underlying mechanism. We hypothesized that MEG8 plays an important role in cardiovascular disease via epigenetic regulation of gene expression. Experiments were performed in human umbilical vein endothelial cells (HUVECs). In vitro silencing of MEG8 resulted in impaired angiogenic sprouting. More specifically, total sprout length was reduced as was proliferation, while migration was unaffected. We performed RNA sequencing to assess changes in gene expression after loss of MEG8. The most profoundly regulated gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), was 5-fold increased following MEG8 silencing. TFPI2 has previously been described as an inhibitor of angiogenesis. Mechanistically, MEG8 silencing resulted in a reduction of the inhibitory histone modification H3K27me3 at the TFPI2 promoter. Interestingly, additional silencing of TFPI2 partially restored angiogenic sprouting capacity but did not affect proliferation of MEG8 silenced cells. In conclusion, silencing of MEG8 impairs endothelial function, suggesting a potential beneficial role in maintaining cell viability. Our study highlights the MEG8/TFPI2 axis as potential therapeutic approach to improve angiogenesis following ischemia.

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The Effect of Grp78 Inhibition on Tissue Factor Gene Expression, Procoagulant Activity, and Factor Xa Generation.

Blood ◽

10.1182/blood.v104.11.1924.1924 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1924-1924

Author(s):

Gourab Bhattacharjee ◽

Jasimuddin Ahamed ◽

Brian Pedersen ◽

Amr El-Sheikh ◽

Cheng Liu ◽

...

Keyword(s):

Gene Expression ◽

Cell Surface ◽

Tissue Factor ◽

Factor Xa ◽

Cell Types ◽

Procoagulant Activity ◽

Coagulation Cascade ◽

Clotting Factor ◽

Tf Gene

Abstract In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.

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Alternate RNA splicing of murine nfkb1 generates a nuclear isoform of the p50 precursor NF-kappa B1 that can function as a transactivator of NF-kappa B-regulated transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8460 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8460-8470 ◽

Cited By ~ 15

Author(s):

R J Grumont ◽

J Fecondo ◽

S Gerondakis

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Rna Splicing ◽

Coding Region ◽

Nf Kappa B ◽

Reading Frame ◽

C Terminus ◽

A Cell ◽

Regulated Gene Expression

The NF-kappa B1 subunit of the transcription factor NF-kappa B is derived by proteolytic cleavage from the N terminus of a 105-kDa precursor protein. The C terminus of p105NF-kappa B1, like those of I kappa B proteins, contains ankyrin-related repeats that inhibit DNA binding and nuclear localization of the precursor and confer I kappa B-like properties upon p105NF-kappa B1. Here we report the characterization of two novel NF-kappa B1 precursor isoforms, p84NF-kappa B1 and p98NF-kappa B1, that arise by alternate splicing within the C-terminal coding region of murine nfkb1. p98NF-kappa B1, which lacks the 111 C-terminal amino acids (aa) of p105NF-kappa B1, has a novel 35-aa C terminus encoded by an alternate reading frame of the gene. p84NF-kappa B1 lacks the C-terminal 190 aa of p105NF-kappa B1, including part of ankyrin repeat 7. RNA and protein analyses indicated that the expression of p84NF-kappa B1 and p98NF-kappa B1 is restricted to certain tissues and that the phorbol myristate acetate-mediated induction of p84NF-kappa B1 and p105NF-kappa B1 differs in a cell-type-specific manner. Both p84NF-kappa B1 and p98NF-kappa B1 are found in the nuclei of transfected cells. Transient transfection analysis revealed that p98NF-kappa B1, but not p105NF-kappa B1 or p84NF-kappa B1, acts as a transactivator of NF-kappa B-regulated gene expression and that this is dependent on sequences in the Rel homology domain required for DNA binding and on the novel 35 C-terminal aa of this isoform. In contrast to previous findings, which indicated that p105NF-kappa B1 does not bind DNA, all of the NF-kappa B1 precursors were found to specifically bind with low affinity to a highly restricted set of NF-kappa B sites in vitro, thereby raising the possibility that certain of the NF-kappa B1 precursor isoforms may directly modulate gene expression.

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